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1 Institute for Virology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 266a, CH-8057 Zurich, Switzerland
2 Clinic for Small Animal Internal Medicine, Dermatology Unit, University of Zurich, Winterthurerstrasse 266a, CH-8057 Zurich, Switzerland
Correspondence
Mathias Ackermann
email{at}vetvir.unizh.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the nucleotide sequence data of CPV3 is DQ295066.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Numerous viruses have been uncovered in EV lesions, but some, such as human papillomaviruses (HPV) 5 and 8, have been associated with cancer development (Nuovo & Ishag, 2000). Similarly, cases in dogs of pigmented plaques (PP) that develop subsequently into squamous-cell carcinoma (SCC) are well documented (Briggs, 1985; Gross & Brimacomb, 1986; Nagata et al., 1995; Stokking et al., 2004; Walder, 1997). Indeed, Nagata et al. (1995) have described conditions similar to EV in dogs, terming these conditions the canine counterpart to human EV. Whilst the classical canine oral papillomavirus (COPV) is able to induce typical warts, neither genomic sequences of COPV nor typical histological lesions thereof have yet been described in association with canine EV (Nagata et al., 1995; Scott et al., 2001). However, Tanabe et al. (2000) amplified a small sequence identified as a part of the papillomavirus L1 gene from such lesions and showed that it differed from its COPV counterpart. Based on preliminary data, including PCR amplification and sequencing of the amplification products (Zaugg et al., 2005), we hypothesized that unknown canine papillomaviruses (CPVs) may exist and that they may be detected in cases of canine EV. Consequently, we extracted DNA from lesions of a dog with suspected EV and amplified circular DNA by the recently described multiple-primed rolling-circle amplification (RCA) method, which has been shown to be useful for the cloning of entire papillomavirus genomes (Dean et al., 2001; Rector et al., 2004a, b, 2005). Indeed, papillomavirus-like DNA was amplified from canine EV before being cloned and sequenced. Sequence determination and analysis resulted in the detection and primary description of a third CPV (CPV3). Moreover, the new isolate meets the majority of criteria needed to declare detection of a novel genus among the papillomaviruses (de Villiers et al., 2004). |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Tissue samples.
Full-thickness biopsies of each type of lesion and adjacent normal skin were taken. One set of biopsies was fixed in 10 % neutral-buffered formalin, embedded in paraffin, sectioned to 4–6 µm slices, mounted onto glass slides and stained with haematoxylin and eosin (H&E) for light microscopy. A second set of tissue was kept at –20 °C until DNA extraction.
Amplification and cloning of genome.
Total DNA from 25 mg tissue (various lesions) was isolated by using a DNeasy extraction kit (Qiagen) according to the manufacturer's recommendations. DNA (1 µl) was used for RCA (Rector et al., 2004b), using a TempliPhi Amplification kit (Amersham Biosciences). The polymerase reaction was primed with protected, random hexamers as supplied in the kit. The protocol supplied by the manufacturer was used, except for a prolonged reaction time for 16 h at 30 °C. Amplified DNA was cloned into the SacI site of pBluescript II-KS+ (Stratagene) by using standard procedures.
Sequence analysis.
The nucleotide sequence of cloned DNA was determined commercially (Microsynth) by cycle sequencing using an ABI 377 sequencer (Applied Biosystems). The primary sequence was then analysed by using the software packages Vector NTI (Informax) and GCG version 10.3 (Accelrys). Program parameters were set to default except where indicated otherwise. Phylogenetic analyses were performed with CLUSTAL_X version 1.83 (Jeanmougin et al., 1998) and PHYLIP version 3.63 (Felsenstein, 2004). A multiple sequence alignment using CLUSTAL_X was calculated with the default parameters (UIB DNA matrix). A bootstrapped (1000 datasets) phylogenetic tree was constructed with PHYLIP, using sequentially the programs SEQBOOT, DNADIST, NEIGHBOR and DRAWTREE. The quality of the tree was evaluated with CONSENSE. The nucleotide sequence data of CPV3 were deposited in GenBank under accession no. DQ295066
[GenBank]
. GenBank accession numbers of sequences used for multiple sequence alignment are shown in Table 1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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exemplifies the histology found in the two main types of lesion. Histology revealed variable acanthosis, ranging from moderate in maculae to pronounced in the ulcerated lesion. Furthermore, microscopic observations included ortho- and sometimes parakeratotic hyperkeratosis, marked hyperpigmentation of all layers of the epidermis, scattered dyskeratotic cells and a marked hypergranulosis with clumped keratohyalin granules. Based on these findings, the diagnosis of canine EV was made. Loss of polarity and nests of basaloid cells were noticed in some sections. The changes were most pronounced in the samples from the ulcerated lesions, where numerous atypia, such as macrocaryosis, anisocaryosis, multinucleated cells or abnormal mitosis, were also present. However, the basal membrane always remained intact. Furthermore, the ulcerated lesion was diagnosed as an in situ SCC. Based on results from a broad-range PCR, suitable for the detection of a wide variety of papillomaviruses, it was hypothesized that this case may be associated with a novel CPV infection (Zaugg et al., 2005).
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).
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). Furthermore, the long control region (LCR), between the end of the L1 gene and the start of the E6 gene, was 566 bp in length. The SacI site used for cloning was located at the end of the ORF encoding the E2 protein. Deduced amino acid sequences of the putative proteins revealed an ATP-dependent helicase motif (GPPDTGKS) (Wilson et al., 2002) in the C-terminal region of E1, two putative metal-binding motifs (CX2CX29CX2C) in the deduced amino acid sequences of E6 and one such motif in E7 (Münger et al., 2004). The amino acid sequences of both structural proteins L1 and L2 were predicted to harbour a basic tail at their C termini. The locations of the predicted ORFs are described in Table 2(a).
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) were detected; seven of them were located in the LCR (positions 7372–7385, 7435–7448, 7541–7554, 7581–7594, 7672–7685, 7731–7744 and 7754–7767) and three were dispersed over the genome (positions 729–742, 3956–3969 and 4915–4928). Within the LCR, a putative origin of DNA replication was identified, consisting of two E2-binding regions (positions 7672–7685 and 7731–7744) flanking an A/T-rich region (positions 7686–7709) and dyad-symmetry repeats (TTGTTGTTAACAACAA; positions 7710–7725). Two polyadenylation consensus sequences (AATAAA) were present at positions 4305–4311 and 7255–7261. The first of them is probably the signal for processing the early protein pre-mRNA and the second the signal for the late pre-mRNA. Thus, all important elements present in a papillomavirus genome were detected. Therefore, the CPV with the presently determined sequence was termed CPV type 3 (CPV3).
Comparison of CPV3 with known PV genomes
In order to address the possible classification of CPV3, alignments were made at the nucleotide sequence level of single ORFs and the translated individual ORFs were compared at either the nucleotide or the amino acid sequence level. First, a phylogenetic tree was constructed based on the multiple sequence alignment of L1 nucleotide sequences (Fig. 4). CPV3 did not group into any of the defined papillomavirus genera, but occupied a position on a side branch to the 
- and 
-papillomaviruses. Apparently, bovine BPV5 (an -papillomavirus) and bovine BPV1 and 2, ovine OvPV1 and 2, European elk EEPV and deer DPV (-papillomaviruses) represent, thus far, the closest relatives.
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). Under these conditions, CPV3's E1 protein sequence was more similar to the homologues of COPV and CPV2 (43 %) than to those of OvPV1 and 2, EEPV and DPV, and BPV1, 2 and 5 (37–42 %). Likewise, the L2, E7 and E6 proteins of CPV3 were more similar to those of COPV and CPV2. However, the L1 protein sequence of CPV3 was less similar to the corresponding protein sequence of CPV2 and COPV (47 and 52 %, respectively) than to BPV1, 2 and 5, OvPV1 and 2, and EEPV (49–55 %). Although only partial sequences of a predicted additional CPV, CPV-PEN, are publicly available (Tanabe et al., 2000), the L1 protein gene of CPV-PEN was compared at the nucleotide and amino acid levels with the corresponding sequences of COPV, CPV2 and CPV3. According to this analysis, CPV3 shared about 70 % sequence identity with CPV-PEN, at both the nucleotide and the amino acid levels, but only about 50 % with the other CPVs (data not shown). Yet, the comparison suggested that CPV3 and CPV-PEN are probably still two distinct viruses.
According to the rules of the International Committee on Taxonomy of Viruses (ICTV), the criteria for the definition of a separate PV genus are fulfilled if <60 % nucleotide sequence identity in the L1 ORF is shared and if complete genomes have >23 %, but <43 % nucleotide sequence identity (de Villiers et al., 2004). To address this issue properly, pairwise sequence alignments covering the L1 ORF and the entire genomic sequences were performed at the nucleotide level. Calculated identities between CPV3 and the corresponding sequences of the most closely related papillomaviruses are listed in Table 2(b). The closest relative to CPV3 was BPV2, with 59 % nucleotide sequence identity in the L1 ORF. Similarly, 58 % identity was found with BPV5 and OvPV2, respectively, whereas the canine PVs retained 56 % (COPV) and 55 % (CPV2) identity in L1. Interestingly, the identities on the entire genome sequences were lower for BPV2 (47 %) and BPV5 (46 %) than for OvPV2 (48 %) and the highest identities to the CPV3 genome sequence were calculated to COPV and CPV2 (50 and 49 %, respectively). Thus, the criteria to declare detection of the prototype of a novel PV genus are met at least partially.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Nagata et al., 1995). In both cases, families or breeds appear to be predisposed to these conditions, even though similar changes may be observed in non-predisposed, immunosuppressed individuals (de Oliveira et al., 2003; Morrison et al., 2002; Nagata et al., 1995; Stokking et al., 2004). Histologically, both conditions exhibit specific changes that are clearly different from the features of classical warts (Nagata et al., 1995; Nuovo & Ishag, 2000). Of note, both conditions seem to be induced by specific papillomaviruses. In humans, EV-associated HPVs, such as HPV 5 and 8, are regularly, but not exclusively, found in EV lesions and are sometimes found in normal skin or skin associated with non-PV-induced conditions (Antonsson et al., 2000; Li et al., 2004). Tanabe et al. (2000) reported the sequencing of a small portion of the genome of one virus (CPV-PEN) found in PP lesions in dogs. The clinical signs described in that report were similar to our observations. Throughout the present work, we amplified a circular DNA with identical restriction-enzyme patterns (data not shown) from several benign lesions, as well as one malignant lesion, of a dog with an EV-like syndrome. The malignant lesion was diagnosed as canine in situ SCC. Following cloning of this DNA, nucleotide sequence determination indicated that it corresponded to a novel papillomavirus, which was termed CPV3.
Further sequence analysis of CPV3 revealed typical elements common to papillomaviruses. In particular, we were able to assign the ORFs potentially encoding L1, L2, E1, E2, E6 and E7. In addition, several recognition sites for E2 DNA binding and two polyadenylation signals were detected. Multiple sequence alignment of the nucleotide sequence encoding L1 allowed the construction of a phylogenetic tree. The criteria of ICTV to be met for declaration of a separate papillomavirus genus were met almost perfectly. Therefore, the novel papillomavirus described in this communication may represent the first member of a novel papillomavirus genus. It occupies a position on the phylogenetic tree in the neighbourhood of the -papillomaviruses (BPV1, 2 and 5) and the -papillomaviruses (OvPV1 and 2, European elk EEPV and deer DPV). The other CPVs included in the phylogenetic analysis were clearly on distinct branches of the tree.
CPV-PEN, a predicted, but hitherto incompletely sequenced, CPV, which has been associated with a clinical disease similar to that associated with CPV3, showed the closest relatedness to CPV3 within the L1 gene sequence (70 % identity). This hypothesis suggests that viruses similar to CPV3 circulate in the canine population and are found in association with EV. Therefore, this new group of papillomaviruses may be of considerable veterinary and, probably, medical interest.
Based on our observations, it would be interesting to compare the entire genome sequence of CPV-PEN with the CPV3 sequence reported here. In particular, the sequences of E6 (and E7) might reveal a connection of the clinical signs and the genome sequences.
In conclusion, we detected and determined the genomic sequence of a novel papillomavirus, which may represent a novel genus within this virus family. The novel virus was found in a dog in association with lesions reminiscent of human EV. Moreover, CPV3 E6 mRNA expression was detected in three different lesions of this dog, but not in its healthy skin (data not shown). Furthermore, sequencing of p53 cDNA obtained from lesions and from normal skin revealed no mutation in comparison with the published canine p53 sequence (GenBank accession no. AB020761 [GenBank] ). These two additional observations sustain the idea of a possible causative association between this novel virus and the disease.
It should be interesting to test in the future whether this condition can be reproduced in experimental animals. If such were the case, a new model for EV could be established on the basis of a natural host species.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 19 June 2006;
accepted 18 August 2006.
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